Bottom Line:
Collectively, the perinuclear reorganization of protein synthesis machinery, the predominant euchromatin architecture, and the active nucleolar transcription could represent compensatory mechanisms in ALS motor neurons in response to the disturbance of ER proteostasis.In this scenario, epigenetic activation of chromatin and nucleolar transcription could have important therapeutic implications for neuroprotection in ALS and other neurodegenerative diseases.Although histone deacetylase inhibitors are currently used as therapeutic agents, we raise the untapped potential of the nucleolar transcription of ribosomal genes as an exciting new target for the therapy of some neurodegenerative diseases.

Affiliation: Service of Neurology, University Hospital Marqués de Valdecilla, Instituto de Investigación Valdecilla (IDIVAL), University of Cantabria , Santander , Spain.

ABSTRACTWe investigated neuronal self-defense mechanisms in a murine model of amyotrophic lateral sclerosis (ALS), the transgenic hSOD1(G93A), during both the asymptomatic and symptomatic stages. This is an experimental model of endoplasmic reticulum (ER) stress with severe chromatolysis. As a compensatory response to translation inhibition, chromatolytic neurons tended to reorganize the protein synthesis machinery at the perinuclear region, preferentially at nuclear infolding domains enriched in nuclear pores. This organization could facilitate nucleo-cytoplasmic traffic of RNAs and proteins at translation sites. By electron microscopy analysis, we observed that the active euchromatin pattern and the reticulated nucleolar configuration of control motor neurons were preserved in ALS chromatolytic neurons. Moreover the 5'-fluorouridine (5'-FU) transcription assay, at the ultrastructural level, revealed high incorporation of the RNA precursor 5'-FU into nascent RNA. Immunogold particles of 5'-FU incorporation were distributed throughout the euchromatin and on the dense fibrillar component of the nucleolus in both control and ALS motor neurons. The high rate of rRNA transcription in ALS motor neurons could maintain ribosome biogenesis under conditions of severe dysfunction of proteostasis. Collectively, the perinuclear reorganization of protein synthesis machinery, the predominant euchromatin architecture, and the active nucleolar transcription could represent compensatory mechanisms in ALS motor neurons in response to the disturbance of ER proteostasis. In this scenario, epigenetic activation of chromatin and nucleolar transcription could have important therapeutic implications for neuroprotection in ALS and other neurodegenerative diseases. Although histone deacetylase inhibitors are currently used as therapeutic agents, we raise the untapped potential of the nucleolar transcription of ribosomal genes as an exciting new target for the therapy of some neurodegenerative diseases.

Figure 3: (A–F) Electron micrographs illustrating the organization of the perinuclear region in motor neurons from control (C) and hSOD1G93A mice (A,B,D–F). (A,B) Low magnification images of chromatolytic neurons showing the euchromatic nuclei with several interchromatin granule clusters (white asterisks) and infoldings of the nuclear envelope. The arrow indicates a Cajal body. Electron-dense cytoplasmic areas, corresponding to local accumulations of free polyribosomes and RER cisterns, appear concentrated at the perinuclear region. Note in (B), a perinuclear cap of polyribosomes at the wrinkled nuclear pole and the presence of large cytoplasmic vacuoles (Vc). (C–F) Detail of the perinuclear region in control (C) and ALS (D–F) motor neurons. The perinuclear cytoplasm exhibits scattered polyribosomes, Golgi cisterns and some mitochondria in the control neuron (C). In contrast, perinuclear accumulations of either concentric arrays of RER cisterns (D) or combinations of free polyribosomes and RER cisterns (E) are observed in an ALS motor neurons. Note in (E,F) that nuclear infoldings contain a great abundance of polyribosomes that fill the depressions of the nuclear envelope. Tangential sections of the nuclear membranes illustrate the great density of nuclear pores at the polyribosome-rich nuclear infoldings [arrows in (F) and inset]. Scale bars: (A,B) = 2.8 μm; (C–E) = 0.9 μm; (F) and inset = 600 nm.

Mentions:
Next, we investigated possible cellular mechanisms involved in neuronal survival under conditions of severe chromatolysis in ALS motor neurons. In particular, we analyzed the reorganization of the RER. Both cytochemical staining with propidium iodide and toluidine blue staining showed the frequent presence of fluorescent or basophilic perinuclear caps enriched in RNA in motor neurons of the hSOD1G93A mice at both presymptomatic and symptomatic stages (Figures 1B,C). The ultrastructural counterpart was the local accumulation of RER cisterns and free polyribosomes in close proximity to the nuclear envelope (Figures 3A,B). While in the majority of control neurons, the perinuclear cytoplasm commonly displayed scattered polyribosomes, Golgi complexes and mitochondria (Figure 3C), two main perinuclear arrangements of the protein synthesis machinery were found in the ALS mouse model. The first consisted of concentric arrays of RER cisterns in close proximity of the nuclear envelope (Figure 3D). The second were perinuclear caps of free polyribosome with some isolated cisterns of RER (Figure 3E). Perinuclear caps were most commonly observed in motor neurons with severe chromatolysis and abnormal accumulations of neurofilaments, and frequently occurred at a wrinkled nuclear pole in which the infoldings of the nuclear envelope were filled with polyribosomes and RER cisterns (Figures 3B,E). Tangential sections of the nuclear envelope at the nuclear infoldings showed high density of nuclear pores and their spatial association with polyribosomes (Figure 3F, inset). The quantitative analysis of the proportion of motor neurons having perinuclear caps of RER revealed a significant increase in ALS mice compared to wild type, with a notable higher frequency in symptomatic than in asymptomatic stages of the ALS (Figure 4E).

Figure 3: (A–F) Electron micrographs illustrating the organization of the perinuclear region in motor neurons from control (C) and hSOD1G93A mice (A,B,D–F). (A,B) Low magnification images of chromatolytic neurons showing the euchromatic nuclei with several interchromatin granule clusters (white asterisks) and infoldings of the nuclear envelope. The arrow indicates a Cajal body. Electron-dense cytoplasmic areas, corresponding to local accumulations of free polyribosomes and RER cisterns, appear concentrated at the perinuclear region. Note in (B), a perinuclear cap of polyribosomes at the wrinkled nuclear pole and the presence of large cytoplasmic vacuoles (Vc). (C–F) Detail of the perinuclear region in control (C) and ALS (D–F) motor neurons. The perinuclear cytoplasm exhibits scattered polyribosomes, Golgi cisterns and some mitochondria in the control neuron (C). In contrast, perinuclear accumulations of either concentric arrays of RER cisterns (D) or combinations of free polyribosomes and RER cisterns (E) are observed in an ALS motor neurons. Note in (E,F) that nuclear infoldings contain a great abundance of polyribosomes that fill the depressions of the nuclear envelope. Tangential sections of the nuclear membranes illustrate the great density of nuclear pores at the polyribosome-rich nuclear infoldings [arrows in (F) and inset]. Scale bars: (A,B) = 2.8 μm; (C–E) = 0.9 μm; (F) and inset = 600 nm.

Mentions:
Next, we investigated possible cellular mechanisms involved in neuronal survival under conditions of severe chromatolysis in ALS motor neurons. In particular, we analyzed the reorganization of the RER. Both cytochemical staining with propidium iodide and toluidine blue staining showed the frequent presence of fluorescent or basophilic perinuclear caps enriched in RNA in motor neurons of the hSOD1G93A mice at both presymptomatic and symptomatic stages (Figures 1B,C). The ultrastructural counterpart was the local accumulation of RER cisterns and free polyribosomes in close proximity to the nuclear envelope (Figures 3A,B). While in the majority of control neurons, the perinuclear cytoplasm commonly displayed scattered polyribosomes, Golgi complexes and mitochondria (Figure 3C), two main perinuclear arrangements of the protein synthesis machinery were found in the ALS mouse model. The first consisted of concentric arrays of RER cisterns in close proximity of the nuclear envelope (Figure 3D). The second were perinuclear caps of free polyribosome with some isolated cisterns of RER (Figure 3E). Perinuclear caps were most commonly observed in motor neurons with severe chromatolysis and abnormal accumulations of neurofilaments, and frequently occurred at a wrinkled nuclear pole in which the infoldings of the nuclear envelope were filled with polyribosomes and RER cisterns (Figures 3B,E). Tangential sections of the nuclear envelope at the nuclear infoldings showed high density of nuclear pores and their spatial association with polyribosomes (Figure 3F, inset). The quantitative analysis of the proportion of motor neurons having perinuclear caps of RER revealed a significant increase in ALS mice compared to wild type, with a notable higher frequency in symptomatic than in asymptomatic stages of the ALS (Figure 4E).

Bottom Line:
Collectively, the perinuclear reorganization of protein synthesis machinery, the predominant euchromatin architecture, and the active nucleolar transcription could represent compensatory mechanisms in ALS motor neurons in response to the disturbance of ER proteostasis.In this scenario, epigenetic activation of chromatin and nucleolar transcription could have important therapeutic implications for neuroprotection in ALS and other neurodegenerative diseases.Although histone deacetylase inhibitors are currently used as therapeutic agents, we raise the untapped potential of the nucleolar transcription of ribosomal genes as an exciting new target for the therapy of some neurodegenerative diseases.

Affiliation:
Service of Neurology, University Hospital Marqués de Valdecilla, Instituto de Investigación Valdecilla (IDIVAL), University of Cantabria , Santander , Spain.

ABSTRACTWe investigated neuronal self-defense mechanisms in a murine model of amyotrophic lateral sclerosis (ALS), the transgenic hSOD1(G93A), during both the asymptomatic and symptomatic stages. This is an experimental model of endoplasmic reticulum (ER) stress with severe chromatolysis. As a compensatory response to translation inhibition, chromatolytic neurons tended to reorganize the protein synthesis machinery at the perinuclear region, preferentially at nuclear infolding domains enriched in nuclear pores. This organization could facilitate nucleo-cytoplasmic traffic of RNAs and proteins at translation sites. By electron microscopy analysis, we observed that the active euchromatin pattern and the reticulated nucleolar configuration of control motor neurons were preserved in ALS chromatolytic neurons. Moreover the 5'-fluorouridine (5'-FU) transcription assay, at the ultrastructural level, revealed high incorporation of the RNA precursor 5'-FU into nascent RNA. Immunogold particles of 5'-FU incorporation were distributed throughout the euchromatin and on the dense fibrillar component of the nucleolus in both control and ALS motor neurons. The high rate of rRNA transcription in ALS motor neurons could maintain ribosome biogenesis under conditions of severe dysfunction of proteostasis. Collectively, the perinuclear reorganization of protein synthesis machinery, the predominant euchromatin architecture, and the active nucleolar transcription could represent compensatory mechanisms in ALS motor neurons in response to the disturbance of ER proteostasis. In this scenario, epigenetic activation of chromatin and nucleolar transcription could have important therapeutic implications for neuroprotection in ALS and other neurodegenerative diseases. Although histone deacetylase inhibitors are currently used as therapeutic agents, we raise the untapped potential of the nucleolar transcription of ribosomal genes as an exciting new target for the therapy of some neurodegenerative diseases.